RESUMO
BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.
Assuntos
Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Metimazol/análogos & derivados , Choque Séptico/imunologia , Choque Séptico/prevenção & controle , Tionas/uso terapêutico , Animais , Modelos Animais de Doenças , Inflamação/imunologia , Masculino , Metimazol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Choque Séptico/metabolismoRESUMO
Thyroglobulin (Tg), a major product of the thyroid gland, serves as a macromolecular precursor of thyroid hormone biosynthesis. In addition, Tg stored in the thyroid follicles is a potent regulator of thyroid-specific gene expression. In conjunction with thyroid stimulating hormone (TSH) and iodide, Tg regulates thyroid follicle function, which is the minimal functional unit of the thyroid gland. In the present study, we show that Tg stimulates growth of FRTL-5 thyroid cells in the absence of TSH, insulin and serum. Unlike TSH, Tg did not increase cellular cyclic AMP (cAMP) levels; rather, the TSH signal counteracted Tg-induced cell growth. A specific inhibitor of A-kinase, H-89, did not modulate the effect of Tg. Tg increased kinase activity of Akt to the same level as TSH, insulin and 5% serum, while LY294002 abolished Tg-induced growth. Interestingly, low Tg concentrations maximized growth-promotion activity and induction of the apical iodide transporter (PDS; SLC26A4), whereas high Tg concentrations suppressed both cell growth and the expression of thyroid-specific genes. These results suggest that a low levels of Tg in the follicular lumen might stimulates cell growth and iodide transport to accelerate the iodide organification process; however, elevated Tg levels in the follicle might then shut down all of these functions.
Assuntos
Proliferação de Células , AMP Cíclico/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/fisiologia , Tireotropina/metabolismo , Animais , Linhagem Celular , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Ratos , Timidina/metabolismo , Tireoglobulina/farmacologia , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/farmacologiaRESUMO
Although it is well known that an excess of iodide suppresses thyroid function and blood flow in vivo, the underlying molecular mechanisms are not fully known. The functional effect of iodide occurs at multiple steps, which include inhibition of sodium/iodide symporter (NIS) expression, transient block of organification, and inhibition of hormonal release. The vascular effect likely involves suppression of the vascular endothelial growth factor (VEGF) gene. In this report, we show that excess iodide coordinately suppresses the expression of the NIS and VEGF genes in FRTL-5 thyroid cells. We also demonstrate that the mechanism of iodide suppression of NIS gene expression is transcriptional, which is synergized by the addition of thyroglobulin. Based on the findings of reporter gene assays and electrophoretic gel mobility shift analysis, we also report two novel DNA binding proteins that responded specifically to iodide and modulated NIS promoter activity. The results suggest that excess iodide affects thyroid vascular function in addition to iodide uptake. This study provides additional insights into the mechanism of action of excess iodide on thyroid function.
Assuntos
Iodetos/farmacologia , Simportadores/genética , Glândula Tireoide/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Iodetos/metabolismo , Ratos , Simportadores/antagonistas & inibidores , Tireoglobulina/metabolismo , Tireoglobulina/farmacologia , Glândula Tireoide/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Quercetin is the most consumed flavonoid present in fruits and vegetables. There has been increased interest in the possible health benefits of quercetin and other flavonoids. Because it is reported that these compounds have some antithyroid properties, we were interested whether, and by what mechanism, quercetin might regulate thyroid cell growth and function. In this report we show that quercetin inhibits thyroid cell growth in association with inhibition of insulin-modulated phosphatidylinositol 3-kinase-Akt kinase activity. Furthermore, quercetin decreases TSH-modulated RNA levels of the thyroid-restricted gene sodium/iodide symporter (NIS). We associated down-regulation of NIS RNA levels with inhibition of iodide uptake at comparable quercetin concentrations and could show that the inhibitory effect of quercetin on NIS RNA levels and iodide uptake is reproduced by inhibitors of the phospholipase-A(2)/lipoxygenase pathway. The specific inhibitor of protein kinase A, H89, only partially inhibited TSH-increased NIS expression and did not reproduce the quercetin effect. The quercetin studies thus reveal that the phospholipase-A(2)/lipoxygenase pathway appears to play an important role in TSH regulation of NIS gene expression, whereas quercetin inhibition of growth appears to involve an effect on insulin/IGF-I-Akt signaling. The data raise the possibility that quercetin may be a novel disruptor of thyroid function, which has potential effects on, or use in, the therapy of thyroid diseases.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Quercetina/farmacologia , Glândula Tireoide/efeitos dos fármacos , Animais , Antitireóideos/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Disruptores Endócrinos/farmacologia , Iodo/metabolismo , Fosfolipases A2/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Simportadores/genética , Simportadores/metabolismo , Simportadores/fisiologia , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiologia , Tireotropina/farmacologiaRESUMO
The increased expression of VCAM-1 on endothelial segments within plaque regions could be used as a target to deliver polymeric drug carriers selectively to sites of atherosclerosis. We probed the hypothesis that polymeric particles conjugated with a ligand for VCAM-1 exhibit selective and avid adhesion to sites of atherosclerosis. Particles made from polystyrene or the biodegradable polymer poly(sebacic acid)-block-polyethylene glycol (PSA-PEG) were conjugated with an antibody to VCAM-1 (alpha-VCAM-1) or IgG (negative control). The particles were injected into the jugular vein of ApoE(-/-) (a murine model of atherosclerosis) or wild type mice and their adhesion to the aorta determined. alpha-VCAM-1 particles exhibited significantly greater adhesion to ApoE(-/-) mouse aorta [32 +/- 5 (mean +/- SEM) particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles] compared to the level of adhesion to wild type mouse aorta (18 +/- 1 particles/mm(2) for polystyrene particles and 6 +/- 1 particles/mm(2) for PSA-PEG particles). Within ApoE(-/-) mice, the alpha-VCAM-1 particles exhibited significantly greater adhesion to the aorta (32 +/- 5 particles/mm(2) for polystyrene particles and 31 +/- 7 particles/mm(2) for PSA-PEG particles) compared to the adhesion of IgG particles (1 +/- 1 particles/mm(2) for polystyrene particles and 2 +/- 1 particles/mm(2) for PSA-PEG particles). Detailed analysis of the adhesion revealed that alpha-VCAM-1 particles exhibited focal adhesion to plaque regions, in particular the periphery of the plaques, within the ApoE(-/-) mouse aorta. Combined the data demonstrate that polymeric particles conjugated with a ligand to VCAM-1 exhibit selective, avid and focal adhesion to sites of atherosclerosis providing strong evidence that VCAM-1 ligand bearing polymeric particles could be used for targeting drugs selectively to atherosclerotic tissue.
Assuntos
Anidridos/metabolismo , Aterosclerose/metabolismo , Adesões Focais/metabolismo , Polietilenoglicóis/metabolismo , Poliestirenos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Análise de Variância , Animais , Anticorpos Monoclonais/metabolismo , Aorta/metabolismo , Portadores de Fármacos/metabolismo , Ligantes , Camundongos , Especificidade por SubstratoRESUMO
We previously reported that hormones important for the normal growth and function of FRTL-5 rat thyroid cells, TSH, or its cAMP signal plus insulin or IGF-I, could transcriptionally suppress constitutive and gamma-interferon (IFN)-increased synthesis of the 90K protein (also known as Mac-2BP). Here we cloned the 5'-flanking region of the rat 90K gene and identified a minimal promoter containing an interferon response element and a consensus E-box or upstream stimulator factor (USF) binding site, which are highly conserved in both the human and murine genes. We show that suppression of constitutive and gamma-IFN-increased 90K gene expression by TSH/cAMP plus insulin/IGF-I depends on the ability of the hormones to decrease the binding of USF to the E-box, located upstream of the interferon response element. This site is required for the constitutive expression of the 90K gene. Transfection with USF1 and USF2 cDNAs increases constitutive promoter activity, attenuates the ability of TSH/cAMP plus insulin/IGF-I to decrease constitutive or gamma-IFN-increased 90K gene expression but does not abrogate the ability of gamma-IFN itself to increase 90K gene expression.
Assuntos
Região 5'-Flanqueadora/genética , Proteínas/genética , Fatores Estimuladores Upstream/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte , Linhagem Celular , AMP Cíclico/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas da Matriz Extracelular , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Interferon gama/farmacologia , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Tireotropina/farmacologia , Transfecção , Fatores Estimuladores Upstream/genéticaRESUMO
High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.
Assuntos
Carcinoma Papilar/genética , Metimazol/análogos & derivados , Metimazol/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Neoplasias da Glândula Tireoide/genética , Receptor 3 Toll-Like/fisiologia , Proteínas Wnt/fisiologia , Carcinoma Papilar/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas/genética , Neoplasias da Glândula Tireoide/patologia , Receptor 3 Toll-Like/genética , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Wnt binding to cell surface receptors can activate a 'canonical' pathway that increases cellular beta-catenin or a 'noncanonical' Ca(++) pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/beta-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of beta-catenin. Transient overexpression of Wnt-1 or beta-catenin in FRTL-5 cells increased active beta-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of beta-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5'-A/T A/T CAAAG-3', -137 to -129 bp on the rat TPO promoter. beta-Catenin overexpression significantly increased complex formation between beta-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the beta-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing beta-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of beta-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression.
Assuntos
Iodeto Peroxidase/genética , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Glândula Tireoide/metabolismo , Transcrição Gênica/fisiologia , Proteína Wnt1/metabolismo , Animais , Northern Blotting/métodos , Western Blotting/métodos , Ciclo Celular/genética , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Expressão Gênica , Iodeto Peroxidase/metabolismo , Ratos , Tireotropina/farmacologia , Transfecção , beta Catenina/genética , beta Catenina/metabolismoRESUMO
We have shown that thyroglobulin (Tg) is a potent autocrine regulator of thyroid-specific gene expression, and proposed that the accumulated follicular Tg within the colloid is a major factor in determining follicular function. In the present report, we examined the effect of Tg on the action of TSH/cAMP and iodine with special focus on the regulation of basolateral and apical iodide transporters; the sodium/iodide symporter (NIS) and the pendred syndrome gene (PDS) by Tg. We show that expression of NIS and PDS are differentially regulated by Tg concentration and exposure time. In addition, we found that PDS gene was induced by TSH/cAMP and iodide in the presence of Tg. Based on these results, we propose a model for the physiological turnover of follicular function that is dynamically regulated by Tg.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Simportadores/fisiologia , Tireoglobulina/fisiologia , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Células Cultivadas , AMP Cíclico/genética , AMP Cíclico/fisiologia , Sinergismo Farmacológico , Feminino , Regulação da Expressão Gênica/genética , Iodo/fisiologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Folículo Ovariano/fisiologia , RNA Mensageiro/genética , Ratos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transportadores de Sulfato , Simportadores/genética , Tireoglobulina/biossíntese , Tireoglobulina/genética , Tireotropina/genética , Tireotropina/fisiologia , Fatores de TempoRESUMO
The expression of certain endothelial cell adhesion molecules (ECAMs) is increased in the vasculature of the inflamed bowel (e.g., colitis), thereby providing an opportunity for targeted drug delivery. We recently demonstrated that biodegradable particles conjugated with ligands to ECAMs exhibit significant selective adhesion to ECAM expressing endothelium. In the present study, we used a murine model of colitis to determine whether poly(lactic acid)-poly(ethylene glycol) particles conjugated with a VCAM-1 ligand (alpha-V) exhibit enhanced adhesion to colitic vasculature. In post-capillary venules of the colon, significantly more alpha-V particles accumulate in colitic mice relative to (i) control mice (i.e., selectivity) and (ii) particles bearing a control ligand (i.e., ligand efficiency). The selectivity and ligand efficiency of alpha-V particles were a function of the total number of particles infused. The highest selectivity observed within our test regime was 3, while ligand efficiency increased linearly with the number of particles injected to a value of 24. This work represents a significant step towards achieving a targeted drug delivery scheme for the treatment of inflammatory bowel disease and indicates that the efficiency of targeting is dependent on the dose regime.
Assuntos
Colite/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Vênulas/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Biotina , Colite/induzido quimicamente , Colite/fisiopatologia , Colo/irrigação sanguínea , Sulfato de Dextrana/administração & dosagem , Portadores de Fármacos/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Poliésteres , Polietilenoglicóis , Polímeros , Poliestirenos , Rodaminas/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.
Assuntos
Expressão Gênica/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Metimazol/análogos & derivados , Metimazol/farmacologia , Receptores de Superfície Celular/genética , Tionas/farmacologia , Tireoidite Autoimune/metabolismo , Viroses/metabolismo , Animais , Quimiocinas CC/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Interferon beta/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , RNA de Cadeia Dupla , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tireoidite Autoimune/patologia , Receptor 3 Toll-Like , Receptores Toll-Like , TransfecçãoRESUMO
Methimazole (methyl-mercapto-imidazole, MMI), a compound used clinically in therapy of Graves' thyroiditis, was found to inhibit development of several autoimmune diseases in animal models. It was suggested on the basis of in vitro data that inhibition is through down-regulation of interferon-gamma (IFN-gamma)-induced expression of major histocompatibility complex class I and class II molecules. Here, we investigate the effect of MMI on experimental autoimmune uveoretinitis (EAU) and study its mechanism(s). Treatment of EAU with MMI administered in drinking water inhibited induction of the disease and associated antigen (Ag)-specific proliferation and cytokine production by draining lymph node cells (LNCs). The treatment was protective only if administered during the first but not during the second week after immunization, suggesting an effect on the induction phase of EAU. It is interesting that MMI inhibited disease in IFN-gamma knockout mice, indicating that the in vivo protective effect is IFN-gamma-independent. Flow cytometric analysis of draining LNCs extracted 5 days after immunization showed that MMI partly to completely reversed the increase in Mac-1(+)/class I(+)/class II(+) cells induced by immunization and reduced the proportion of B7-1 and CD40-positive cells, suggesting a deficit in the Ag-presenting cell (APC) population. APC from untreated mice largely restored antigen-specific proliferation of MMI-treated LNCs. We suggest that MMI inhibits EAU at least in part by preventing the recruitment and/or maturation of APC, resulting in reduced generation of Ag-specific T cells.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Antitireóideos/farmacologia , Doenças Autoimunes/prevenção & controle , Proteínas do Olho , Metimazol/farmacologia , Uveíte/imunologia , Uveíte/prevenção & controle , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Antitireóideos/imunologia , Doenças Autoimunes/tratamento farmacológico , Citocinas/análise , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Interferon gama/farmacologia , Linfonodos/citologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/efeitos dos fármacos , Metimazol/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retinite/tratamento farmacológico , Retinite/imunologia , Retinite/prevenção & controle , Proteínas de Ligação ao Retinol/administração & dosagem , Proteínas de Ligação ao Retinol/imunologia , Uveíte/tratamento farmacológicoRESUMO
Increased major histocompatibility complex (MHC) class I gene expression in target tissues may be relevant to the pathogenesis of autoimmune diseases. In this study, we questioned whether high glucose levels might increase MHC class I levels and thereby contribute to autoimmune complications. We used thyrocytes in continuous culture, because there is an increased incidence of autoimmune thyroiditis in type 2 diabetics and because transcriptional regulation of MHC class I is well studied in these cells. Northern analysis and flow cytometry showed that 20 and 30 mM D-glucose up-regulated MHC class I expression and that the glucose effect was additive to and independent of interferon-gamma. The effect was specific, because L-glucose did not modify class I expression. The glucose acted transcriptionally, requiring both enhancer A and a cAMP-response element-like element located in the hormone-sensitive region of the MHC class I 5'flanking region. These elements are different from those activated by interferon-gamma. High glucose levels increase formation of the MOD-1 complex with enhancer A; MOD-1 is a heterodimer of fra-2 and the p50 subunit of NF-kappaB. Both TSH and insulin are required for full expression of the glucose activity in thyrocytes. The glucose effect is partially blocked by wortmannin, suggesting involvement of the PI3K signal system. The data support the possibility that high serum glucose levels in type 2 diabetic patients may increase MHC class I levels in target tissues and contribute to autoimmune complications of the disease.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I/genética , Glucose/farmacologia , Interferon gama/fisiologia , Glândula Tireoide/metabolismo , Animais , Northern Blotting , Células Clonais , Citometria de Fluxo , Insulina/fisiologia , Luciferases/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Ratos , Glândula Tireoide/efeitos dos fármacos , Tireotropina/fisiologia , TransfecçãoRESUMO
Here we report the cloning of the rat 90K, a homolog of the mouse cyclophilin C-associated protein/mouse adherent macrophage and human 90K. The protein is constitutively expressed by FRTL-5 thyrocytes, and its levels are modulated by TSH, insulin/IGF-I, and gamma-interferon. Transfection of the cells with 90K cDNA or exposure to purified 90K resulted in a significant increase of the expression of major histocompatibility complex class I but not class II antigens. An increased expression of 90K was obtained after viral infection or introduction into the cells of fragments of viral, bacterial, or mammalian double-strand polynucleotides. The increase was sequence independent, not CpG mediated, and associated with the expression of molecules characterizing antigen-presenting-cell phenotype. The present data along with results from previous studies suggest that 90K plays an important role in the maintenance of an appropriate level of immune response.
Assuntos
DNA/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Hormônios/farmacologia , Interferon gama/farmacologia , Polinucleotídeos/farmacologia , Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte , Linhagem Celular , Clonagem Molecular , Proteínas da Matriz Extracelular , Herpes Simples/metabolismo , Humanos , Dados de Sequência Molecular , Polinucleotídeos/genética , Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
We performed an electrophysiological study to investigate ion transport of pendrin and thereby understand the pathogenesis of Pendred syndrome. Using pendrin-transfected COS-7 cells, we could show that pendrin transports both iodide and chloride measured as voltage-dependent inward and outward membrane currents. Chloride in the culture medium, [Cl-]o, was efficiently exchanged with cytoplasmic iodide, [I-]i, under physiological concentrations, indicating that pendrin is important for chloride uptake and iodide efflux. Although exchange of iodide in the medium, [I-]o, with cytoplasmic chloride, [Cl-]i, was observed, a significantly high concentration of iodide (10 mm) was required. In addition, either iodide or chloride was required on both sides of the cell membrane for the anion exchange activity of pendrin, indicating that iodide and chloride activate the exchange activity of pendrin while they are transported. The present study further supports that pendrin is responsible for the iodide efflux in thyroid cells where intracellular iodide concentration is high and that the general function of pendrin in other tissues is to transport chloride through exchange with other anions.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloretos/farmacocinética , Iodetos/farmacocinética , Proteínas de Membrana Transportadoras , Animais , Ânions/metabolismo , Células COS , Citoplasma/metabolismo , Humanos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Transportadores de Sulfato , TransfecçãoRESUMO
The Pendred syndrome gene encodes a 780-amino acid putative transmembrane protein (pendrin) that is expressed in the apical membrane of thyroid follicular cells. Although pendrin was shown to transport iodide and chloride using Xenopus laevis oocytes and Sf9 insect cells, there is no report using mammalian cells to study its role in thyroid function. We show here, using COS-7 cells and Chinese hamster ovary cells transfected with expression vectors encoding sodium iodide symporter or human Pendred syndrome gene cDNA and by comparison with studies using rat thyroid FRTL-5 cells, that pendrin is an iodide-specific transporter in mammalian cells and is responsible for iodide efflux in the thyroid.
Assuntos
Proteínas de Transporte/fisiologia , Iodetos/farmacocinética , Proteínas de Membrana Transportadoras , Glândula Tireoide/metabolismo , Animais , Transporte Biológico/fisiologia , Células CHO , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Cloretos/farmacologia , Cricetinae , Humanos , Iodo/farmacocinética , Radioisótopos do Iodo , Ratos , Transportadores de Sulfato , Simportadores/fisiologia , Glândula Tireoide/citologiaRESUMO
Differences in the epitopes of thyroid-stimulating antibodies (TSAbs) from patients with untreated Graves' disease were compared with long-term response to antithyroid drugs. Epitopes were measured using Chinese hamster ovary cells transfected with wild-type human TSH receptor (TSHR) and two receptor chimeras, wherein TSHR residues 9-165 or 90-165 had been substituted with comparable residues of the LH/chorionic gonadotropin receptor. Of 159 patients studied, 52 (32.7%) exhibited positive TSAb activity with one or both chimera lines (heterogeneous group), and 107 (67.3%) had no activity with either (homogeneous group). Independent of all other parameters, patients with heterogeneous epitopes responded more favorably to oral antithyroid drugs than patients with homogeneous epitopes (65.4% vs. 41.9%, P = 0.011: estimated odds ratio by logistic regression, 2.17). Although most clinical parameters were not different at presentation, significant differences in the size of goiters, total T(3) concentrations, and titers of TSH-binding inhibitory Igs were evident in the successfully treated group (n = 80) by comparison to the group of patients whose treatment failed (n = 79). Alone, these three parameters did not predict outcome; however, when either of these parameters were considered together with epitope heterogeneity, predictability of a positive therapeutic response was increased to nearly 80%. Thus, the presence of TSAbs with a heterogeneous epitope in a patient with Graves' disease is not only associated with a favorable response to antithyroid drug treatment, it may help predict the response to treatment when the patient is initially seen.
Assuntos
Antitireóideos/uso terapêutico , Epitopos , Variação Genética , Doença de Graves/tratamento farmacológico , Doença de Graves/imunologia , Imunoglobulinas Estimuladoras da Glândula Tireoide/imunologia , Animais , Células CHO , Linhagem Celular , Quimera , Cricetinae , Previsões , Humanos , Imunoglobulina G/imunologia , Receptores do LH/genética , Receptores da Tireotropina/genética , Resultado do TratamentoRESUMO
Expression of the Pendred syndrome gene (PDS/Pds) is thought to be responsible for the iodide transport in the thyroid as well as the formation and function of the inner ear. Its mRNA is also expressed in the kidney and placenta. We report here that PDS and its encoded protein (pendrin) are also expressed in the endometrium. The RNA levels of rat PDS in the endometrium and kidney were much higher than those of the thyroid, opposite of the pattern of RNA expression in humans. In human endometrium, pendrin localization changed from the basal to apical surfaces of the epithelium during progression of the menstrual cycle. This suggests a possible role for pendrin in cationic ion transport required to maintain the physiological function of the endometrium. Since there is no evidence of endometrial abnormalities in patients with Pendred syndrome, it suggests the existence of a compensatory mechanisms for pendrin's function in the uterus.
Assuntos
Proteínas de Transporte/genética , Endométrio/fisiologia , Expressão Gênica , Proteínas de Membrana Transportadoras , Animais , Epitélio/metabolismo , Feminino , Humanos , Ciclo Menstrual/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transportadores de Sulfato , Distribuição TecidualRESUMO
In normal human subjects a small proportion of peripheral blood T-cells simultaneously express both CD4 and CD8 differentiation antigens. In this study we characterized a subset of CD4+ clones, from a healthy donor, that is specific for the thyrotropin receptor (TSHR) and that showed cells co-expressing the CD8 receptor. To address whether the expression of the CD8 receptor on the cell membrane was associated to differences in the physiology of the T-cells, we isolated, from the same clone, CD4 single positive (SP) cells from those co-expressing CD4/CD8 receptors (DP cells) and stimulated them in vitro with antigen presenting cells (APC) carrying TSHR. The results demonstrated that CD8 co-expression has a profound effect on the physiology of T helper (Th) cells. In comparison to cells expressing the CD4 receptor alone, DP T-cells showed: (1) increased proliferation; (2) higher and more sustained release of free Ca2+ in the cytosol, under stimulus; (3) lower levels of IL-2 and IL-4 released in the supernatants; (4) increased amounts of IFN-gamma released.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cálcio/metabolismo , Citocinas/biossíntese , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Humanos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.